In recent years, there has been an increase in demand for pH color-changing materials. These materials can visually communicate signals to people by connecting pH changes with color information. Embedding pH indicators into fibers to create flexible color-changing materials is an effective way to develop daily wearable products. For the stability of the indicator and the indirect contact of the indicator with the human body, it is usually necessary to encapsulate it in capsules. In this study, different pH indicators  were mixed into a wide-domain pH color-changing indicator and encapsulated with ethyl cellulose (EC) by the flash nanoprecipitation (FNP) method using a new-type droplet-shaped confined impinging jet mixer. The effects of flow rate, core-to-wall ratio, and mixed solution concentration on the formation of the nanocapsules were investigated. In addition, the morphology, particle size, size distribution, dispersion stability, and encapsulation efficiency were systematically studied. At a core-to-wall ratio of 1:2, a mixed solution with a concentration of 6 mg/mL and a feed flow rate of 40 mL/min produced nanocapsules with an average particle size of 141.83 ± 0.98 nm and a PDI of 0.125 ± 0.01. Furthermore, a zeta potential with a range of −31.83 ± 0.23 mV and an encapsulation efficiency of 75.20 ± 1.72% were observed at 1:2 core-to-wall ratios. It was concluded that the color of the nanocapsules continuously changed from yellow to green and green to blue when the pH range was increased from 3 to 10. The color-changing nanocapsules were then embedded into sodium alginate hydrogel fibers, resulting in the same color-changing trend (pH 3–10) as that obtained for the nanocapsules. This study can be useful for the pH monitoring of various body fluids, such as wound exudate, urine, and sweat. The pH color-changing materials change their color according to the pH value and can lead to sensitive, controlled, and visible environmental response signals in a nondestructive manner. Developing smart color-changing materials with good flexibility and mechanical stability is considered an important new smart textile material with a wide range of applications in different areas. Generally, pH color-changing materials are mainly detected by combining pH color-changing substances with novel chemical substrates through doping, adsorption, chemical bonding, and coating . SchuereCompared to natural pH indicators, chemical reagents are more sensitive to discoloration, easier to access, and lower in cost. However, the direct use of a single chemical pH indicator may lead to poor stability, contact toxicity, and a narrow indication domain ,5,6,7, wThe flash nanoprecipitation (FNP) method is a novel processing technique used for the preparation of nanoparticles that is based on the kinetically controlled principle of turbulent fluid mixing in chemical engineering . GeneralHydrogel fibers have the functional properties of high water content, high elasticity, and stimulus response to materials. These properties of hydrogel fibers contribute to materials with a high specific surface area and easy weaving fibrous properties . In addiConsidering all the facts together, this study addresses the mixing of three pH indicators  to create a new pH indicator, which can broaden the pH color-changing domain. Then, a wide range of pH color-changing nanocapsules were prepared by a droplet-shaped CIJ mixer with the mixed indicator acting as the core material and ethyl cellulose as the wall material. In the pH color-changing material field, these nanocapsules are firmly bonded with substrates in various forms to develop pH color-changing-related products. Furthermore, the potential applications of the nanocapsules in sodium alginate hydrogel fibers were also explored in this study. The nanocapsules created in this study can be useful for monitoring body fluids, such as sweat, wound exudate, and urine.Ethyl cellulose  and polyvinyl alcohol 1788  were purchased from Aladdin . Thymol Blue (TB) and Bromocresol Green (BCG) were obtained from Macklin . Bromocresol Purple (BCP) was purchased from Yonghua Chemical Co., Ltd. . Ethanol (AR) was obtained from Titanchem . pH buffer solution (pH = 3–10) was purchased from Jiangbiao Testing Technology Co., Ltd. . Deionized water (from the Milli-Q water purification system) was used in all experiments. All chemicals were used without any further purification.v/v) and stirred magnetically at 600 rpm for 3 h at room temperature until the indicators were completely dissolved in the solution. The resulting solution was used for the preparation of a mixed solution with a total concentration of 6 mg/mL and a core-to-wall ratio of 1:2. In addition, mixed solutions with mass concentrations of 8 and 10 mg/mL and core-to-wall ratios of 1:1 and 1:3 were separately prepared. A PVA solution with a mass concentration of 0.1 mg/mL was prepared by mixing 0.01 g PVA in 100 mL deionized water at room temperature at 600 revolutions per minute (RPM) for 1 h until the PVA was fully dissolved.The pH indicators TB, BCG, and BCP were mixed in a 1:1:1 ratio. TB-BCG-BCP (0.2 g) and EC (0.4 g) were dissolved in 100 mL of ethanol solution . The suspension of nanocapsules was dropped on a silicon wafer. When the silicon wafer was dry, the nanocapsules were subjected to conductive adhesive and sprayed with gold for 90 s. The scanning electron microscope voltage was set at 3.0 kV.−1.Pure TB-BCG-BCP, EC, and EC/TB-BCG-BCP mixtures and discolored nanocapsules (NCs) were tested separately using FTIR spectroscopy, on the NICOLET5700 iS5 . Specimens were prepared by the potassium bromide press method and scanned within the wavelength range of 500–4000 cmThe prepared nanocapsule suspension was centrifuged by the centrifuge H3-18KR , and the precipitates were taken for dilution. The particle size, dispersity index (PDI), and particle size distribution of the nanocapsules were measured using the Nano ZS90 Malvern laser particle size analyzer . The measurements were repeated three times, and the results were averaged.The zeta potential of the nanocapsule suspension was tested using the Nano ZS90 Malvern Zeta Potentiometer . Each sample was tested in triplicate.TB-BCG-BCP solutions were prepared at different mass concentrations and subjected to UV full-spectrum scanning. The wavelengths were set from 350 to 600 nm to find the maximum absorption value in the visible range.The absorbance of the centrifuged TB-BCG-BCP supernatant was measured at the maximum absorption wavelength using the UV-Vis spectrophotometer, Specord S600 . The mass concentration of the supernatant was calculated according to the standard curve equation. The encapsulation efficiency was measured according to Formula (1):The method for quantifying color information is based on the HSV color model, where the HSV color space describes the specific color through the three components of hue (H), saturation (S), and value (V) ,17. In tThe color changes of the nanocapsules in different pH environments were photographed by a smartphone camera (64 MP) in natural light. The color information was then quantified by identifying and extracting the H-value of the image using Adobe Photoshop (CC 2017) software.All experiments were performed in triplicate. The average and standard deviation of the particle size, zeta potential, and encapsulation efficiency results were calculated with the Microsoft Excel (2016) program.2H5O-) aggregated inward, thus encapsulating the hydrophobic core [The mixed solution of TB-BCG-BCP and EC (positive solvent) and the aqueous PVA solution (antisolvent) are injected at the same flow rate into both inlets of the CIJ mixer. The positive and antisolvent collided and mixed in a confined and narrow space within milliseconds. With the rapid increase in the proportion of antisolvent, the solubility of TB-BCG-BCP reached supersaturation and precipitated to form hydrophobic cores, while the hydrophilic group (-OH) of EC extended toward the aqueous phase and the hydrophobic group , the characteristic absorption peak of the -OH group is at 3457 cm−1. The characteristic absorption peak of the benzene ring group of nanocapsules was noted at 1588 cm−1. However, the characteristic absorption peak of the -CH3 group was observed at 1451 cm−1, and the symmetric and asymmetric stretching vibrations of the -SO2- group were observed at 1161 and 1344 cm−1, respectively.The infrared spectrum of EC (green line) reveals that the peak at 3481 cm−1. The characteristic absorption peaks of TB-BCG-BCP in the range of 1100–1600 cm−1 were significantly weakened. However, the characteristic absorption peak of TB-BCG-BCP was located in the spectrum of the mixture of EC and TB-BCG-BCP (red line). This demonstrates that TB-BCG-BCP was well encapsulated by EC.Compared to the infrared spectra of pure TB-BC-BCP and EC, the spectrum of the nanocapsules (black line) showed an enhancement of the -OH group at approximately 3400 cmThe core-to-wall ratio (1:2) and the mixed solution concentration (6 mg/mL) were kept constant. The particle size, PDI, and size distribution of the nanocapsules at different flow rates are displayed in The flow rate is an important parameter during nanoparticle synthesis using a CIJ mixer. A higher flow rate results in a higher level of supersaturation, leading to a higher nucleation rate and facilitating the production of smaller and more homogeneous nanoparticles . HoweverThe flow rate (40 mL/min) and the mixed solution concentration (6 mg/mL) were kept constant. The particle size, PDI, and size distribution of the nanocapsules at different core-to-wall mass ratios are shown in The flow rate (40 mL/min) and the core-to-wall ratio (1:2) were kept constant. The zeta potential test results of nanocapsule suspensions with different antisolvents are shown in As shown in 2 = 0.99989).The UV absorption spectra of different concentrations of TB-BCG-BCP-water standard solutions were examined and are shown in The encapsulation efficiency was calculated using the method introduced in 2.3.5, and the results are shown in As shown in As shown in For color responsiveness, 400 μL of the nanocapsule suspension was added dropwise to 150 μL of a buffer solution with an increasing pH (pH 3–10) . Figure Alginate hydrogel fibers are inherently flexible and can be shaped into irregular geometries, making them easier to use in various applications . They ar2 coagulation bath at a rate of 10 mL/min. The hydrogel fibers were washed with deionized water and then collected.We mixed the 3% w/v SA solution with the nanocapsule suspension in a 2:1 volume ratio and connected the syringe containing the mixture to an 18 G needle using a silicone tube. The syringe pump squeezed the syringe mixture into a 2% w/v CaClThe hydrogel fibers were divided into eight equal lengths and put into separate bottles with different buffer solutions in relation to pH (pH 3–10) . Figure 2 solutions. The embedded loading of the nanocapsules and the transparency of the hydrogel allow for successful color-changing sensing under acidic and alkaline conditions, which shows the potential application in the field of medical dressings, paper diapers, and sports straps to monitor the pH values of wound exudate, urine, and sweat, respectively.In this study, wide-domain pH color-changing nanocapsules were prepared by the flash nanoprecipitation method with a new-type droplet-shaped confined impinging jet mixer. Mixing the three indicators at an optimum mixing ratio of 1:1:1, the pH color-changing domain of the mixed indicators extended to pH 3–10. The excellent preparation process for the nanocapsulation was as follows: the core-to-wall ratio was 1:2, the concentration of the mixed solution was 6 mg/mL, and the feed flow rate was 40 mL/min. Under these conditions, the nanocapsules obtained were as follows: the average particle size was 141.83 ± 0.98 nm, the PDI was 0.125 ± 0.01, the zeta potential was −31.83 ± 0.23 mV, and the encapsulation efficiency was 75.20 ± 1.72%, with good storage and dispersion stability. The nanocapsules exhibited a continuous color change from yellow to green and then to blue when the pH values changed from 3 to 10. In addition, the nanocapsules can be mixed directly with sodium alginate solutions to form hydrogels or hydrogel fibers by ionic cross-linking with CaCl


Ischemic stroke (IS) is a serious neurological disease that largely results in long-term disability and death. Extensive evidence has indicated that the activation of inflammation and ferroptosis significantly contribute to the development of IS pathology. However, the underlying molecular mechanism remains unclear. In this study, we aimed to identify potential biomarkers associated with IS through the construction of a competing endogenous RNA (ceRNA) network and to investigate the possible inflammatory and ferroptosis-related molecular mechanisms.We identified 178 differentially expressed target messenger RNAs (DETmRNAs) associated with IS. As revealed through enrichment analysis, the DEmRNAs were mainly enriched in the inflammatory signaling pathways and also related to ferroptosis mechanism. The CIBERSORT algorithm showed immune infiltration landscapes in which the naïve B cells, naïve T cells, and monocytes had statistically different numbers in the cerebral infarction group compared with the control group. A ceRNA network was constructed in this study involving 44 long non-coding RNAs (lncRNAs), 15 microRNAs (miRNAs), and 160 messenger RNAs (mRNAs). We used the receiver operating characteristic (ROC) analysis to identify three miRNAs , one mRNA (TLR4), and one lncRNA (NEAT1) as the potential key biomarkers of the ceRNA network. The key mRNA and lncRNA were shown to be highly related to the ferroptosis mechanism of IS. The expression of these key biomarkers was also further validated by a method of quantitative real-time polymerase chain reaction in SH-SY5Y cells, and the validated results were consistent with the findings predicted by bioinformatics.Our results suggest that the ceRNA network may exert an important role in the inflammatory and ferroptosis molecular mechanisms of IS, providing new insight into therapeutic IS targets.The online version contains supplementary material available at 10.1186/s12864-023-09163-1. Globally, stroke remained the second-leading cause of death and the third-leading cause of long-term disability , 2. StatDespite advances in understandings of the causes and treatments of ischemic stroke (IS), its exact molecular mechanisms are not yet entirely clear. Thus, a better understanding of the pathological processes of IS may provide novel therapeutic targets and methods for IS, thus improving prognosis . MountinAs an important class of pervasive genes, several studies have found that non-coding RNAs (ncRNAs) play a key role in IS-induced neuroinflammation and iron overload –18. MicrLong non-coding RNAs (lncRNAs) are also a kind of non-coding RNA with more than 200 nucleotides in length that regulate target gene expression by interacting directly with mRNAs as a transcriptional regulator, or by acting as competing endogenous RNAs (ceRNAs), for miRNAs , 22 and At present, the ceRNA axis has become a research hot spot, and the interactions among lncRNAs, miRNAs, and mRNAs may provide valuable insight into the molecular mechanisms of IS and present novel biomarkers or targets for rehabilitation treatment . In the https://www.ncbi.nlm.nih.gov/geo/), which is a public functional genomics data repository. IS-related datasets were retrieved using the keyword “stroke of Homo sapiens” (organisms). We included the gene expression profiling of the whole or peripheral blood of IS patients or control samples. Four datasets with reliable sample sources were downloaded by using the GEO query package [http://www.zhounan.org/ferrdb) [Expression profile data associated with stroke were obtained from the GEO database ( package  of R sof package  (v3.44.0/ferrdb) , includiP < 0.05, |log 2-fold change (FC)| > 0.5 were selected as cut-off criteria in identifying DEmRNAs and DEmiRNAs by previously reported methods [We analyzed the expression matrixes of miRNA, lncRNA, and mRNA microarray. Differential expression analysis of the miRNA dataset of GSE110993 was provided in the GEO datasets. Other microarrays were reannotated using the platform annotation file. The probe annotation of this series was based on Entrez Gene ID. The data were quantile normalized, and the differential expressed miRNAs (DEmiRNAs), mRNAs (DEmRNAs), and lncRNAs (DElncRNAs) between the IS and control samples were identified using R’s limma package (v3.52.0) . The P < methods , 28–30,  methods , 32. The methods .http://bioinformatics.PSB.ugent.be/webtools/Venn), a Venn diagram was drawn to determine the DEmiRNAs shared between the two miRNA datasets (GSE110993 and GSE117064). Second, the online databases were utilized to pair lncRNA–miRNA and mRNA–miRNA, respectively. The target mRNAs of the DEmiRNAs were predicted using the miRWalk , which is a comprehensive database for predicting miRNA target genes and verifying miRNA binding sites [https://starbase.sysu.edu.cn/), which is a database that specifically records the interactions between miRNAs and lncRNAs [First, using the Venn diagram online drawing tool  and the DElncRNAs that overlapped with the target lncRNA as the differentially expressed target lncRNAs (DETlncRNAs). Finally, the DETlncRNAs–DEmiRNAs and DEmiRNAs–DETmRNAs interactors were subsequently linked to constructing the ceRNA network using the Cytoscape software (v3.8.0) , which ihttps://david.ncifcrf.gov/home.jsp) [P < 0.05 was set as a significant filtering criterion in the enrichment analysis. The results of the enrichment analysis were visualized by the R package ggplot2 package (v3.3.6) [Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to predict functional roles and other shared features of the above DETmRNAs based on the online tools of the Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources . P < 0.0(v3.3.6) .string-db.org/) [Given that proteins are the biomacromolecules that execute functions in our bodies, the online Search Tool for the Retrieval of Interacting Genes (STRING) database   was appldb.org/)  was useddb.org/) , 40.https://cibersortx.stanford.edu/) and parallel (v4.2.0), e1071 (v1.7–9), and preprocess Core (v1.58.0) packages in R for analysis. The relative percentages of 22 immune cell infiltration matrices and the immune infiltration distribution results in each sample of cerebral infarction were visualized using a bar plot. The ggplot2 package (v3.3.6) [P < 0.05 was accepted as the filtering cut-off value.CIBERSORT  is an an(v3.3.6)  in R wasThe diagnosis prediction of IS addressed a classification problem  in this study. We analyzed the classification and prediction efficiencies of the selected top 10 hub miRNAs ranked by the degree in the ceRNA network in the external validation cohort (GSE95204). The pROC package (v1.18.0)  in R wasWe verified the diagnostic performance of the key mRNAs in the discovery dataset (GSE58294) and validation series (GSE16561) and the key lncRNAs in the discovery dataset (GSE198710) and validation series (GSE102541), using the pROC package  to disti2 incubator. The medium was changed every other day, and the cells were subcultured about twice a week. In order to lyse the adherent cells, they were grown to 80% confluence and rinsed with phosphate buffer saline before trypsinization. Thereafter, the SH-SY5Y cells were seeded in 6-well plates for 24 h and randomly divided into the control and model groups.The SH-SY5Y cells are human neuroblastoma cells sharing similar properties with neurons in morphology, neurochemistry, and electrophysiology , which aAn application of Hydrogen peroxide (H2O2) was used to mimic neural injury . The H2O2O2 to SH-SY5Y cells. The cells were seeded into 96-well plates and treated with different concentrations of H2O2 ranging from 0 to 5000 μM for 6 h. Then 10 μ l CCK-8 regent in 90 μ l medium was added to each well and incubated at 37 C for 1 h. The optical density was measured at 450 nm using a microplate reader .The cell viability was detected using a cell counting kit-8  for testing the toxicity of H-ΔΔCt method. GAPDH and RNU6B (U6) were set as the internal control. The primer sequences are listed in Additional file The relative expression level of key miRNAs, lncRNAs, and mRNAs was verified using qRT-PCR. Total RNA was extracted using the NucleoZol RNA reagent  from the SH-SY5Y cells of the control and model groups according to the manufacturer’s instructions. The RNA was eluted in 10 μL DEPC-treated water, quantified by Thermo Scientific NanoDrop 2000 spectrophotometer , and stored at − 80 °C. On one hand, the PrimeScript RT reagent kit with a gDNA Eraser  was used for reverse transcription  of the mRNAs. On the other hand, the PrimeScript RT Master Mix  was used to synthesize cDNA  for the miRNA. We performed qRT-PCR using TB Green Premix Ex Taq II , according to the specifications provided by the manufacturer, on the Applied Biosystems 7500 fluorescence quantitative PCR instrument . The relative expression levels of the RNAs were calculated using the 2t-test was used to estimate normally distributed variables, while the Mann-Whitney U test was used to compare non-normally distributed variables. The ROC curves were visualized, and the corresponding AUC values of the ROC curves were calculated using the pROC package in R. For each study, P < 0.05 was considered a significant difference.The statistical analyses were processed using the R software (version 4.2.0) or SPSS 23.0 statistical software . The independent samples Student’s The result of the differential expression analysis for GSE110993 was downloaded directly from GEO datasets. The expression matrixes of the other six datasets were obtained after pre-processing identically by background correction and normalization. We used R software to perform differential expression analysis on the gene expression matrix between stroke patients and healthy controls. In our study, a total of 140 DEmiRNAs in GSE110993, 430 DEmiRNAs in GSE117064, 334 DEmRNAs in GSE58294, and 2398 DElncRNAs in GSE198710 were identified. The results are shown in the volcano plots , we found that 23 DEmiRNAs were changed consistently. These results were visualized by Venn diagrams  terms include cellular response to sorbitol, positive regulation of nitric oxide biosynthetic process, positive regulation of nitric-oxide synthase biosynthetic process, positive regulation of interleukin-8 production, and so on. The cellular component (CC) reveals the terms of cytosol, mitochondrion, endoplasmic reticulum membrane, endoplasmic reticulum, and so on. The molecular function (MF) terms are mainly associated with protein binding, protein kinase activity, ATP binding, MAP kinase activity, and so on. KEGG pathway analysis has shown that inflammatory-related functions, such as the Toll-like receptor signaling pathway and NF-kappa B signaling pathway, were significantly enriched. Detailed information on GO analysis and KEGG pathway analysis were shown in Fig. GO and KEGG examinations on the 178 DETmRNAs were conducted. Both GO and KEGG enrichment analyses indicated that inflammatory genes may be crucial for IS development. We set adjusted Using the STRING database, a total of 120 PPI interaction logs were predicted from the 178 DETmRNAs used to construct the PPI network Fig.a. The DeWe obtained the immune cell infiltration matrix and the results of immune infiltration distribution in IS samples. The bar plot clearly showed the contents of varied subpopulations in each individual Fig. a. The viThe diagnostic performances of the top 10 hub miRNA to distinguish patients with IS and controls were appraised via ROC analysis in the validation cohort GSE95204). We identify miR-103a-3p , miR-140-3p , and miR-17-5p  as key miRNAs , that is, the concentration of a drug that inhibits cell growth by 50% in different treatments . In thisP-value< 0.05) , 1 lncRNA (NEAT1), and 1 mRNA (TLR4) were verified using qRT-PCR. As predicted, the relative expression level of these biomarkers in the model groups was significantly different from the control groups  in the post-stroke ischemic area, which can exacerbate post-stroke brain injury and promote cell death . NeutropFerroptosis is a recently identified type of iron-dependent cell death . A plethOur study does have several limitations. First, the sample size of each dataset was small, and therefore, additional high-throughput sequencing experiments with larger samples are needed to confirm the findings. Second, this study contained datasets from different testing platforms, which may have biased our results from those generated using data from the same platform. Third, this study only used ROC analysis and qRT-PCR to demonstrate the diagnostic and prognostic values; therefore, future mechanistic research should involve more experiments. Finally, the ceRNA mechanism should be validated by dual luciferase reporter gene assays and in vitro and in vivo knockdown or overexpression.In conclusion, this study constructed an IS-related ceRNA network and identified several signaling pathways associated with IS inflammation and biomarkers related to inflammation and ferroptosis, which are consistent with current pathological knowledge of the disease. We believe that this study provides a new understanding of the molecular mechanisms of IS pathogenesis and that the key RNAs identified herein may become new therapeutic targets for IS rehabilitation strategies.Additional file 1. The 396 human ferroptosis-related genes.Additional file 2. Primer sequence for qRT-PCR.Additional file 3. Interactions of lncRNA-miRNA and miRNA-mRNA.Additional file 4. ROC curve of key miRNAs in the validation set:(a) has-miR-140-3p; (b) has-miR-103a-3p; (c) has-miR-17-5p; (d) has-miR-18a-5p; (e) has-let-5d-5p; (f) has-miR-7f-5p; (g) has-miR-652-3p; (h) has-miR-92a-3p.


Their predictive power was statistically confirmed by univariate analysis and was especially enhanced when combining them. Thus, in the present study, all three scores, simple, fast and, if necessary, also without cytogenetic information, represent significant tools for fitness evaluation before intensive treatment of elderly AML.In elderly patients with acute myeloid leukemia (AML), non-relapse mortality (NRM) after intensive chemotherapy and, if necessary, allogeneic hematopoietic stem cell transplantation (HSCT) depends on an accurate initial patient fitness assessment. We determined three scores in 197 patients at diagnosis and subsequently investigated the patients’ respective overall survival with each fitness score assignment. The G8-score, the HCT-CI and the AML-score for complete remission (CR) were able to significantly separate “fit” from “unfit” patients, p = 0.008. In univariate Cox models, the predictive role for OS was confirmed: for the G8-score , the HCT-CI  and the AML-score , the latter was subsequently used to verify the cohort. In the multivariate Cox model, the results were confirmed for the G8-  and AML-score . Of interest, when combining the scores, their prediction capacity was significantly enhanced, p < 0.001. Conclusions: The G8-, the HCTCI and the AML-score represent valid tools in the frailty assessment of elderly AML patients at diagnosis.Background: Accurate assessment of elderly acute myeloid leukemia (AML) patients is essential before intensive induction chemotherapy and subsequent allogeneic hematopoietic stem cell transplantation. In this context, we investigated the capacity of three scores for frailty prediction. Methods: At diagnosis, 197 patients were clinically evaluated for appropriate treatment intensity. In parallel and independently, the G8-score, the Hematopoietic Stem Cell Index (HCT-CI) and the AML-score for CR were determined for each patient and analyzed with respect to overall survival (OS). Results: The G8-score and the HCT-CI were able to significantly separate “fit” from “unfit” patients, <0.001 and  The age of 60, although arbitrary and not evidence-based, is internationally held to be the cut-off point between “older” and “younger” patients with acute myeloid leukemia (AML) . The proThere are several reasons for these outcome rates. First of all, age itself is an independent, negative prognostic factor in AML . The higIt is thus essential to accurately assess patients’ fitness at diagnosis to determine whether they are eligible for intensive chemotherapy and, if necessary, allogeneic hematopoietic stem cell transplantation (HSCT). The indicators that usually rely on performance status, chronological age, and the presence of comorbidities may not be adequate measures for assessing of the heterogeneity of elderly AML. Several studies have devised and proposed scores aiming to predict NRM and patient outcome.In this context, Palmieri and colleagues retrospectively validated the Ferrara criteria to classify patients’ fitness for intensive chemotherapy . The preMoreover, fitness scores from different settings, such as the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) ,16,17,18The aim of the present study was to investigate the fitness assessment capacity of two scores, namely the HCT-CI and the G8 score ,21. ThesAs shown by the CONSORT diagram in Acute promyelocytic leukemia patients were excluded. Each patient was evaluated at diagnosis by a hematologist according to general performance status, a clinical examination, and, when available, echocardiography and spirometry. Each case was then referred to a multidisciplinary board for discussion of the most appropriate treatment intensity and type. The same board would then be involved in any later re-evaluation of cases after intensive induction chemotherapy before proceeding to allogeneic HSCT or other changes in treatment. Meanwhile, each patient’s fitness was assessed using three scores: (i) a regional geriatric G8 score ,21, (ii)The G8 score, initially developed to identify newly diagnosed cancer patients for more comprehensive geriatric assessment ,21, consFurther, the HCT-CI, a tool for capturing comorbidities before allogeneic HSCT, was calculated for each patient . The HCTwww.AML-score.org (accessed on 1 April 2022) [Lastly, the AML-score for CR was calculated for each patient. Standard clinical and laboratory variables  as well as the variables, if present at diagnosis, for cytogenetic and molecular risk  were inserted into an online form at il 2022) . The datIn order to remove the possibility of bias, the scores remained blinded to the multidisciplinary board responsible for making the final decision about the most appropriate therapy regimen for each patient. The study was conducted in accordance with the Declaration of Helsinki and the guidelines for Good Clinical Practice and approved by the Institutional Ethic Committee (00244/2020 in 20 May 2020).Cytogenetic categorization into favorable-, intermediate- and adverse-risk groups as well as treatment response followed the criteria published on behalf of the European Leukemia Network . PatientThe primary endpoint was OS, defined as the time from the start of intensive chemotherapy to death from any cause. OS was investigated either by the Kaplan-Meier method  or by the Cox proportional hazards model . The predictive role on OS was tested for the above-cited scores: HCT-CI- (≥3 vs. 0–2), G8 score (≤14 vs. >14) and the AML-score for CR . In the latter case, its optimal cut-point was estimated by the maximally selected rank statistics, an outcome-oriented method providing an AML-score for the CR cut-off value that corresponds to the most significant relation with OS .p-values were obtained using the two-sided exact method at the conventional 5% significance level. All data as of May 2022 were analyzed by the R 4.2.0 MaxStat package .Patients’ characteristics were tested with Fisher’s exact test for categorical variables and the Mann–Whitney test for quantitative ones, describing them as median . All A total of 197 patients were evaluated by the hematologist and then successively by the multidisciplinary board. Seventy-seven of them were declared to be clinically unfit for intensive chemotherapy and prescribed demethylating agents, low-dose cytarabine or best supportive care (BSC). The remaining 120 patients were evaluated as clinically fit for intensive chemotherapy and treated according to the “7+3”  or “FLAI2 d1-6, etoposid 80 mg/m2 and cytarabine for 6 h 1 g/m2 d1-6 i.v.) or the FAM- protocols. Of note, most of the intensively treated patients were classified as WHO high-risk patients (59.5%) with ELN-adverse cytogenetics (45.8%) [A subgroup of 27 patients (22.5%) required dose adjustments for age and comorbidities. To compare similarly treated patients, only the 120 patients undergoing intensive chemotherapy were included in the analyses. In cases where CR was achieved, consolidation therapy proceeded with intermediate-dose cytarabine and early allogeneic HSCT. In cases of primary induction failure (PIF), remission was re-challenged with the MEC-(mitoxantron 6 mg/m (45.8%) .Nearly half of the patients, n = 54 (44.6%) developed grade III/IV toxicities after induction chemotherapy, mainly consisting of infectious events, new-onset atrial fibrillation and bleeding complications . A totalAfter intensive induction chemotherapy, complete remission was achieved in 73/120 (60.8%) patients, while 40 (33.3%) were refractory. Twenty-five (20%) of the PIF patients were able to complete a re-induction cycle, 13 (10.8%) of whom achieved a second CR . The resWith a median follow-up (FU) of 38.7 months (IQR: 27.2–75.1), the median OS of the clinically fit defined patients undergoing intensive chemotherapy was 15.0 months (IQR: 6.7–50.5). Of note, the median OS of the 77 clinically unfit declared patients who received demethylating agents or BSC was 3.5 months (IQR: 1.2–9.6).p = 0.147  had a median OS of 20.1 months (IQR: 6.7—not reached) compared to 66–70-year-old patients  with 14.6 months (IQR: 7.4—not reached) and ≥71-year-old patients  with a median OS of 12.6 months (IQR: 6.1–22.7),  = 0.147 .p < 0.001  fit from 55 (45.8%) unfit patients. G8-fit classified patients had a significantly longer median OS of 20.7 months (IQR: 11.5—not reached) than the G8-unfit classified ones with a median OS of 7.8 months (IQR: 4.4–20.6),  < 0.001 A. So did = 0.008 B.p = 0.006  and Study Alliance Leukemia (SAL), based on two large cohorts of elderly AML patients in the same setting. Krug et al. hypothesized that CR achievement is a prerequisite for long-term survival . Followi = 0.006 C. The hip < 0.001 . For this reason, a cut-point selection analysis using the maximally selected rank statistics was performed . The ide < 0.001 D; the mep < 0.001), whereas the HCT-CI resulted in a HR of 1.78  and the AML-score for CR resulted in a HR of 4.07 .Next, to analyze the ability of each of the three scores to independently predict OS, a series of univariate Cox models was performed. The predictive role of all three scores for OS was highly significant. The G8 score resulted in a HR of 2.33  and AML . The four survival models are reported in While all three scores had a significant impact on OS, when analyzed by a series of univariate Cox models, the multivariate one confirmed the OS prognostic role only for G8 .After having shown the prognostic capacity of each single score, the next step was to analyze their combined capacity in a hypothetical model. Four subgroups were created by creating a first subgroup containing only patients with no unfavorable scores and adding another unfavorable score to each successive subgroup, such that the fourth subgroup contained patients with three unfavorable scores. The OS of each of the groups was then compared. The combined capacity of the three scores proved to be highly statistically significant. As the number of unfit scores increased, the OS in the respective subgroup decreased. Patients with all three unfavorable scores (15.8%) had a median OS of 6.2 months (IQR: 2.7–20.7), while patients with two unfavorable scores (34.2%) had a median OS of 8.2 months (IQR: 5.4–17.5). Patients with just one unfavorable score (38.3%) had a median OS of 16.2 months (IQR: 11.5—not reached), while for patients with no unfavorable scores (11.7%), the median OS was not reached  patients as unfit and 16 (20.8%) as fit, while the HCT-CI assessed 41 (53.2%) as fit and 36 (46.8%) patients as unfit. Of the clinically unfit patients defined by the AML score for CR, using the same cut-off value of 63.2% as a binary variable identified 74 (96.1%) patients as unfit and 3 (3.9%) patients as fit.In the present study, the G8 score and HCT-CI were calculated for each patient at diagnosis before intensive chemotherapy and resulted in an assignment of fit or unfit. Stratification by the scores was conducted in parallel and independently from the clinical treatment decision. Consequently, patients’ OS was associated with fitness assignment. Each single score, when considered independently, significantly distinguished fit from unfit patients: the median OS of the G8- as well as of the HCT-CI-fit patients was significantly longer than that of those categorized as unfit. Given that the scores were applied to a clinically selected patient cohort, the AML-score for CR, also determined at diagnosis, was applied to “validate” the cohort. Indeed, in the clinically selected population, fitness prediction by the AML-score in terms of overall survival also proved to be highly significant. The median OS for the AML-score-fit patients was not reached, while the median OS for the AML-score-unfit patients was 12.1 months.The G8 score as well as the HCT-CI are validated tools developed for patient selection in different clinical settings. The G8 questionnaire, with a mean completion time of 5 min is a fast, simple, and feasible screening tool for identifying older cancer patients at risk of life-threatening events. The ONCODAGE Prospective Multicenter Cohort Study showed good sensitivity and independent prognostic value of the G8 score on 1-year survival of oncological patients . In the In addition to the G8 score, the HCT-CI also significantly distinguished fit from unfit patients in terms of OS prediction. The HCT-CI was initially developed by Sorror and colleagues as a tool for capturing pre-transplant comorbidities and was used in predicting the outcome of allogeneic HSCT . SubsequIt was found that not just the individual scores independently significantly distinguished fit from unfit patients, but that the combination of the G8 score, the HCT-CI and the AML-score for CR also resulted in highly significant results, as analyzed in a hypothetical model for OS prediction. The combination of the AML-score with multi-parametric geriatric assessments was recently investigated . Min andHowever, in the cohort of Min et al., a relatively high 58% of patients underwent allogeneic HSCT, as opposed to 27.4% in our study. This may be due to the stringent baseline selection for intensive chemotherapy in the Min cohort; patients with active infection or impaired organ function were excluded from intensive chemotherapy. In patients with highly active disease, organ dysfunction might result in seriously compromised performance status at diagnosis, not reflecting the patients’ usual fitness . TherefoKRAS, ASXL1, RUNX, SETBP1, DNMT3A and CSF3R, are proving to be more discriminative than the ELN 2017 classification in risk stratification [NPM1-mutated cases [IDH1-mutated AML [Cytogenetics are currently considered the most powerful prognostic factor, although recent trials have reported their limitations in elderly AML ,30. The fication ,34,35. Ted cases  or ivosiated AML . Even trated AML  or venetated AML ,40 are cated AML ,42.Although several studies have shown that waiting for cytogenetics ,44 does Our study has several limitations. The selection of OS instead of early death as the primary endpoint may be objected to when addressing patients’ fitness for intensive chemotherapy. In our center, the early mortality rate at 60 days (5.8%) was considerably low. As already underlined by Krug and colleagues, because CR after intensive induction chemotherapy is a prerequisite for long-term survival in AML, the AML-score for CR was used as a surrogate marker for OS prediction .As a single-center study, only patients who were considered fit for intensive induction chemotherapy were included. This selection bias was addressed by analyzing the selected population according to the AML-score established in a larger cohort in the same setting. Another limitation may rest in the two different induction chemotherapy protocols (“7+3”-or FLAI protocol) used, although several studies have shown that the chosen intensive treatment regimens at the time of patient acquisition do not greatly affect outcome in elderly AML patients . The preIn conclusion, the G8 score, the HCT-CI and the AML-score for CR are quick to obtain even without cytogenetics and represent significant tools in the baseline fitness assessment of elderly AML before intensive chemotherapy. Their predictive power for OS is enhanced even when using them in combination. Given that confirmation data is lacking, an individual assessment of each case remains mandatory. Prospective trials will investigate their role in the context of novel intensive chemotherapy regimens incorporating targeting molecules, especially within the recently published ELN 2022 classification .


In contrast, the presence of disease at surgery characterizes a prognostically heterogeneous group of patients. Here, we challenged circulating microRNAs (miRNAs) at the end of neoadjuvant therapy as potential prognostic biomarkers in the NeoALTTO study.The absence of breast cancer cells in surgical specimens, i.e., pre-operative weekly trastuzumab for 6 weeks followed by the addition of weekly paclitaxel for 12 weeks; post-operative FEC for 3 cycles followed by trastuzumab up to complete 1 year of treatment) were randomized into a training (n= 54) and testing (n= 72) set. RT-PCR-based high-throughput miRNA profile was performed on plasma samples collected at the end of neoadjuvant treatment of both sets. After normalization, circulating miRNAs associated with event free survival (EFS) were identified by univariate and multivariate Cox regression model.Patients treated within the trastuzumab arm  in the entire study cohort. By resorting to a multivariate Cox regression model we found a statistical significant interaction between the expression values of miR-194-5p and pCR status (p.interaction =0.005) with an estimate Hazard Ratio (HR) of 1.83 (95%CI 1.14- 2.95) in patients with pCR, and 0.87 (95%CI 0.69-1.10) in those without pCR. Notably, the model including this interaction along with the abovementioned 3-circulating miRNA signature provided the highest discriminatory capability with a C-statistic of 0.67 (95%CI 0.58-0.76).Circulating miRNAs are informative to identify patients with different prognosis among those with heterogeneous response after trastuzumab-based neoadjuvant treatment, and may be an exploitable tool to select candidates for salvage adjuvant therapy. Neoadjuvant therapy is progressively replacing adjuvant therapy and is emerging as a new standard of care for early-stage HER2-positive and triple negative breast cancer . PrimariThe efficacy of additional treatments in HER2-positive breast cancer patients not achieving a pCR after neoadjuvant therapy is being investigated in several studies , and hasn = 154), trastuzumab (n= 149), or their combination (n= 152) for 6 weeks, followed by paclitaxel for 12 weeks. Surgery was performed within four weeks from the last paclitaxel dose. After surgery, patients received fluorouracil, epirubicin, and cyclophosphamide for 3 cycles and continued the same anti-HER2-targeted agent of the neoadjuvant phase to complete 52 weeks of treatment. NeoALTTO primary endpoint was pCR; secondary endpoints included EFS, defined as the time from randomization to first event. As reported . The Cqreference was computed according to the overall mean approach , were separated within 2 hours of collection into plasma aliquots by centrifugation (2000-3000g for 15 minutes at room temperature) and stored at −80°C until assayed at the central biobank of Vall d’Hebron University Hospital . Plasma samples were shipped to Fondazione IRCCS Istituto Nazionale dei Tumori for RNA isolation as already reported . The BF approach .In the training set the association between circulating miRNAs levels  and event free survival (EFS) was assessed by resorting to a univariate Cox regression model. Event-free survival was defined as time from randomization to first event  . The relOut of 149 patients treated within the NeoALTTO trastuzumab arm, 126 (85%) had evaluable circulating miRNA profile at the time of surgery (study cohort). The median age at breast cancer diagnosis was 48 years . Most of the patients had clinical T2 (64%) and ≥N1 tumors (72%). Almost half (47%) had ER-positive tumors. pCR was observed in 31% of cases; a total of 40 events were reported at a median follow-up of 6.7 years . Patients of the study cohort were randomized in a training (n= 54) and testing set (n= 72). No difference in clinico-pathological characteristics was observed between training and testing sets Table 1,In the training set, 23 circulating miRNAs were significantly associated with EFS by univariate analysis , a totalNoteworthy, this 3-circulating miRNA signature was evenly distributed among clinico-pathological variables Figure 1As pCR is a driver of EFS in the NeoALTTO and other neoadjuvant studies, we next analyzed the prognostic value of miRNA according to pCR status. For this purpose, the expression levels of each of the 132 selected miRNAs detected in the study cohort was evaluated thought a multivariate Cox regression model with pCR status and the first-order interaction between miRNA and pCR. We found circulating miR-194-5p with a statistically significant interaction term at alpha level of 0.01 (p.interaction =0.005). The HR estimate in patients with pCR was 1.83 (95%CI 1.14-2.95), and 0.87 (95%CI 0.69-1.10) in those without pCR. For exploratory purposes, an analysis was performed by adding the interaction terms together with the corresponding main effects (circulating miR-194-5p and pCR) to the 3-circulating miRNA signature. The highest prognostic performance in terms of C-statistics was observed for the complete multivariate model Table 3.Not all early-stage breast cancer patients with residual disease after neoadjuvant therapy have a poor prognosis; on the other hand, some patients achieving a pCR eventually relapse. Therefore, biomarkers are needed to properly identify patients at risk who are ideal candidates for additional post-surgical therapies. Using high-throughput analysis of plasma samples collected within a prospective randomized trial , we hereFirstly, we identified a circulating signature composed of miR-185-5p, miR-146a-5p and miR-22-3p able to discriminate among patients treated with trastuzumab-based therapy with different prognosis. The functions of miRNAs composing our prognostic signature have been associated with tumor-related processes , and response to treatment. Specifically, miR-185-5p seems to act as a tumor suppressor in cancer progression and spreading, at least in central nervous system , and gasSecondly, the 3 circulating miRNA signature ensures a C-statistic of 0.62 (0.53-0.7), instead of 0.58 (0.48-0.67) of the model including clinico-pathological variables, age, stage, estrogen receptor status and pCR. Thus, only three circulating markers assessed at a single time point offer a similar if not superior discriminatory capability of different variables assessed on patient, and primary tumor at baseline or after treatment. Furthermore, the discriminatory capability of the 3-circulating miRNA signature remained significant when clinico-pathological variables were included in multivariate analysis. All these findings support the development of our 3 circulating miRNA signature as a parsimonious and independent prognostic tool.Thirdly, despite the favorable prognostic effect of invasive disease eradication, a few of patients with pCR eventually relapse , 10. Tumvice versa. In view of developing a clinically usable assay, the identification of a limited number of reference miRNAs as well as assay-oriented step(s) should be considered, as described by Verderio et al. (This study has some limitations. First, due to its retrospective nature, biological data were available in most but not all patients included in the NeoALTTO trastuzumab arm. Secondly, the analyzed sample size and the number of breast cancer events prevented sub-group analysis. Finally, the number of patients included in the analysis limits the impact of our findings. Therefore, the results of this study need a further confirmation in a much larger patient population, and with an extended follow-up period. Lastly, the prognostic value of TILs was not evaluated in this study. This would be interesting to be assessed in future studies. In addition, studied patients received additional adjuvant chemo-, ± endocrine- and trastuzumab-therapy and we are unaware of the impact of these treatments on miRNAs and o et al. . As regao et al. , for exao et al. . The defIn conclusion, we have identified a 3 circulating miRNA signature able to differentiate among patients with distinct prognosis after trastuzumab-based neoadjuvant therapy, and the unique circulating-miR-194-5p associated with recurrence after pCR attainment, which warrant further investigation in additional studies. If confirmed, these miRNAs and their possible mechanisms of action could aid the development of new post-neoadjuvant strategies for high risk breast cancer patients. Furthermore, given the absence of tumor tissue in patients attaining a pCR, our results reinforce liquid biopsy as a promising tool to quantify and analyze residual breast cancer burden beyond pathological findings and to predict tumor evolution for post-operative therapy personalization.The raw data supporting the conclusions of this article will be made available by the authors upon reasonable request.The studies involving human participants were reviewed and approved by Ethics Committee of Fondazione IRCCS Istituto Nazionale dei Tumori. The patients/participants provided their written informed consent to participate in this study.SDC conceived of the presented idea. SDC, CMC, SP and PV contributed to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript. VC and MS verified the analytical methods. All authors contributed to the article and approved the submitted version.


The created hydrogel has been shown to be biocompatible by toxicological testing of the network. Based on the findings, the created polymeric nexus proved promising for pH-dependent localized and regulated delivery of Diloxanide furoate.This research was carried out to create a pH-responsive polymeric system for the targeted drug delivery of Diloxanide furoate. It relied on sodium alginate (Na-Alg) and Carbopol 934P as building blocks. Using an aqueous free radical polymerization method, SCH1-SCH12 was created with varying polymer, MAA, and MBA input ratios. Positive outcomes were seen in the swelling and release profiles at higher pH levels. Hydrogel formation, as well as component compatibility, thermal stability, and Diloxanide furoate loading, were all validated by instrumental characterization. A drug loading percentage of 83.56% was determined, with the swelling reaching 743.19%. For the formulation with MBA, the gel fraction was 94.58%. The release of diloxanide furoate increased to 91.77% at neutral pH. The formulation containing Carbopol 934P provided the highest mucoadhesion force (3993.42 dynes/cm Over the last several years, controlled localized drug delivery carriers have supplanted more traditional delivery carriers, leading to more widespread treatments. This curiosity stems from the clinical requirement of precisely dosing controlled drugs to specific biological regions for maximum pharmacological effect. Novel biodegradable polymers have made a localized distribution of medication to particular areas a viable method for lowering systemic toxicity . Over thUsing polymers like pectin, dextran, and Na-Alg is a promising strategy for intestinal-specific delivery [Na-Alg). Enzymes in the digestive tract break those down. Hydrogels, made of polymer networks, show promise as controlled delivery methods. Hydrogels, three-dimensional polymeric networks, may be made from either synthetic or natural substances. They can be shaped easily because of their flexibility and high-water content. Hydrogels formed from these polymers have outstanding mucoadhesive properties due to the pendant groups’ adhesion to mucous membranes and the polymers’ pH responsiveness thanks to the pendant groups’ acidic or basic nature, allowing for tailored distribution. The stomach’s pH causes it to contract when the pH is low and expand when the pH high, like in the intestine. Because of their superior control over drug release, hydrogels have become an attractive approach for administering medications without unwanted side effects . AccordiVarious “conventional” chemical techniques may be used to create hydrogels. There are both one-step methods, such as polymerization and the parallel cross-linking of multifunctional monomers, and multi-step methods. The advancements in polymer science have allowed the synthesis of polymeric networks to be controlled on the molecular level, allowing for the design of networks with specific properties. Cross-linking density, biodegradability, mechanical strength, and other aspects of biological and chemical responses to stimuli may be altered by using various polymers, monomers, etc., and by using alternative methods .Na-Alg may be found as a white to yellowish fibrous or granular powder with almost no discernible odor. It dissolves easily in water but not in chloroform, ether, or alcohol. It leads to the formation of very thick colloidal solutions in water . Na-Alg Carbopol 934P is a synthetic hydrophilic biocompatible, biodegradable stable polymer that increases the delivery system’s toughness and flexibility. The cosmetic and pharmaceutical sectors have taken advantage of its excellent swelling and thickening characteristics. Regarding release patterns, hydrophilicity and the presence of -COOH groups are crucial since they impart equilibrium swelling . Such poAcrylates, such as acrylic acid, hydroxyethyl methacrylate, methacrylic acid, and others, may be included in the formulation of a carrier to make it pH-responsive. Methacrylic acid-based networks are sensitive to their surroundings. Because its carboxylic groups are ionized into carboxylate ions at the basic pH of GIT, MAA is crucial, as it modulates swelling behavior .N-methylacetamido) furan-2-carboxylate, and it is a part of the biopharmaceutical classification system (BCS) class II. Solubility is low in ether, water, and alcohol but high in chloroform [Diloxanide furoate (DF) is phenyl 4-, and 99.7% methanol were among the materials used. For the acquisition of NaOH, KH2PO4, NaH2PO4, and NaCl, we reached out to Dae-Jung Chemical Company in Gyeonggi-do, Korea. All chemicals used were of a pure and undamaged analytical grade. The distilled water was just made in a research facility at the University of Lahore’s Faculty of Pharmacy.Thankfully, diloxanide furoate was donated by Adamjee Pharmaceuticals (DF). The St. Louis, MO, USA headquarters of Sigma-Aldrich Co., was the source of our materials. Na-Alg, Carbopol 934P, 99.9% Methacrylic acid (MAA), benzyl peroxide, 99.9% By altering the polymer ratios to the monomer to cross-linker, we created twelve distinct formulations (SCH-1 to SCH-12) using the free radical polymerization approach . A measuThen, the mixture was sonicated for 5–10 min to eliminate any remaining oxygen and air bubbles. Right away, the concoction was poured into test tubes. Aluminum foil-wrapped glass vials were submerged in a 45°C water bath. The first hour saw a hold at this temperature, followed by 50 degrees Celsius for 2 hours, 55 degrees for 2 hours, 60 degrees for 16 h, and 65 degrees for 2hours. After 24 h, the test tubes were removed from the water bath, brought to room temperature, and the hydrogel cylinders were removed by fracturing the test tubes with the wooden back of the test tube holder. The hydrogel cylinders were washed for several minutes under running water to flush out any glass splinters that may have made their way inside. The hydrogel was rolled out, and then cut into 8 mm discs and cleaned three times in a 70:30 ethanol: water solution to remove any residue or unreacted monomer. The discs were air-dried at room temperature before being heated to 45 degrees Celsius in a hot air oven .The cross-linking of carbopol 934P with the terminal carbon of cross-linker MBA produced a sandwich structure. Also, the oxygen atom of methacrylic acid (MAA) was hydrogen-bonded by the hydrogen atom of Carbopol 934P, creating an electrostatic link between the two molecules. The sodium alginate was able to form a connection with the MAA after having a water molecule removed. The potential chemical bonding and electrostatic interactions in the synthesized Na-Alg/CP-co-poly (methacrylate) hydrogel are further elaborated in w/v) DF solution, we weighed them precisely on a digital scale . The DF solution comprised 70% methanol and 30% phosphate buffer (pH = 7.4). When the discs were submerged in water, we let them swell until they reached a constant diameter. After being soaked in the drug solution, the discs were removed and washed quickly in water to remove any remaining drug molecules. Before being heated at 45 degrees Celsius, the discs were allowed to air dry. The drug-loaded hydrogel’s mass was determined as follows:DW = Hydrogel disc with DF loaded, at final weight; dW is the initial weight of hydrogel discs before loading [DF was introduced into the hydrogel discs by swelling-assisted diffusion. Before immersing the dried hydrogel discs in a 1%  for each formulation was determined using Equation (2) .(2)EquiW0 and iW, respectively.Sol-gel analysis was used to calculate the number of unreacted components in the hydrogel network. Hydrogel discs were fabricated and then divided into pieces with a diameter of between 2 and 4 mm, after which they were dried at ambient temperature and then in an oven at 45 °C until a constant weight was attained (W0). The dried materials were boiled for four hours in the Soxhlet apparatus to remove the compounds of interest. After 4 h, we dismantled the machine, transferred the pieces to a glass petri dish, and let them air dry for 24 h before drying them at 45 °C . We dete−1 range [Hydrogel network structural cross-linking and component compatibility were analyzed using Fourier-transformed infrared spectroscopy (FTIR). The spectra of pure DF, Na-Alg, carbopol-934P, MAA, and the produced hydrogel were recorded using an FTIR spectrophotometer . Crushed samples were combined with 12 mm pellets of KBr (Merck IR spectroscopy grade) to acquire spectra in the 4000–400 cm−1 range .The DSC and TGA measurements verified the glass transition temperatures and the thermal stability of the created network compared to pure components at elevated temperatures. Differential Scanning Calorimetry (DSC) analysis was conducted using TA instruments . Two- to five-milligram samples were heated in an oven at temperatures between zero and four hundred degrees Celsius. During heating, 20 mL/min of nitrogen flow and 20°C/min of heat were employed .The materials were analyzed using a thermogravimetric analyzer . After passing through sieve no. 40, the hydrogel samples (0.5–5 mg) were transferred to an aluminum pan, an open microbalance that is dynamically heated from 0 to 500 degrees Celsius at a rate of 20 degrees per minute in a nitrogen environment .X-ray diffraction analysis was used to establish whether the medication, polymer, and network were crystalline or amorphous. The analysis was performed at room temperature using an X-ray diffractometer . Scanning a range of 2 from 10°–70°, peaks were obtained while processing the samples using copper Kα as the radiation source .To ascertain the elemental composition and loading of DF, the EDX spectra of the developed unloaded and DF-loaded networks were recorded using an EDX spectrophotometer  .The generated hydrogel’s surface morphology was studied using scanning electron microscopy (SEM). To dry the chosen discs under a vacuum, they were placed on metal stubs and secured with double-sided adhesive tape before being placed in the vacuum chamber. A sputter coater was used to apply a layer of gold 10 mm thick, which was then examined at several magnifications .Using the USP dissolving equipment II and buffers with pH 1.2 and 7.4, an in vitro drug release investigation of the DF-loaded polymeric networks was performed. The discs were submerged in 900 cc of dissolving media, and the paddle speed was maintained at 50 rpm throughout the process. Throughout the experiment, the medium was kept at a constant 37 ± 2 °C. At 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, and 72 h, aliquots of 5 mL were taken from the vessel’s center and replaced with an equal amount of newly produced buffer to keep the medium at constant sink conditions. After filtration, the samples were analyzed using a UV Visible Spectrophotometer set to a maximum wavelength of 259.8 nm [2 value was used to choose the most appropriate model. If n is less than 0.45, the transport is characterized by Fickian diffusion; if it is between 0.45 and 0.89, the result is non-Fickian or anomalous transport; and if it is more than 0.85, then the result is super case II transport. The following are the equations used in these models:F: the released drug fraction of DF in the hydrogel at time t ; oK, K1, and HK are ZO, FO, and Hig models rate constants, respectively.Mt/M∞ corresponds to the amount of DF released at time t, kpK to the rate constant, and n to the release exponent [The optimal model and mechanism of DF release from the designed hydrogels were identified by processing the dissolution data using a DD solver add-in, Microsoft Excel-based application. We accomplished this by considering various mathematical kinetic models, such as the First Order (FO), Zero Order (ZO), Higuchi (Hig), and Korsmeyer-Pappas (KP) models. The n value was utilized to evaluate the drug release pattern, while the Rexponent .Experiments on the Ex-vivo Mucoadhesion of Na-Alg/CP-co-poly(methacrylate) hydrogels were conducted utilizing modified beam balance equipment, as illustrated in The intestinal tissue of a newly slaughtered rabbit was dissected, turned upside down, and washed in a NaCl solution of 0.9% before being placed in phosphate buffer saline at 37 degrees Celsius for half an hour. The intestinal mucosa receptors were two V-shaped metal caps placed on glass vials. The intestinal mucosal membranes (4 cm in diameter) were glued to the outside of the vials using cyanoacrylate adhesive. The height adjustment pan (H) and the balance were attached to the first vial, while the second was connected to the third vial (B). The left pan of the beam has counterweights hung from it for balance (W). Hydrogel discs (D) were positioned between the mucosal tissues of the two vials shown in  by puttim is the mass in grams needed to separate two mucosal membranes, g is the acceleration due to gravity, and A is the area of tissue removed before attachment.The vials were eventually able to be removed due to the steadily increasing weights. The formula used to determine the detachment force as a measure of mucoadhesion was as follows:The Organization for Economic Co-operation and Development (OECD)’s guidelines for acute oral toxicity testing were used to evaluate the synthesized hydrogels. The research procedures were reviewed by the Institutional Research Ethics Committee (IREC) in the School of Pharmacy at the University of Lahore. It approved the proposal in a letter dated 2020–02-28, including the notification number IREC-2020-28. A week was sufficient to acclimatize 12 healthy albino rabbits since there was a 12-h day/night cycle, enough food and water, and an ambient temperature of 22–30°C. Each of the two groups of rabbits consisted of six animals. They constituted the treatment and control groups created out of the sample pool. The control group had access to unlimited food and drink. The treatment group rabbits were given 2–4 g of powdered hydrogel formulation per kilogram of body weight after 12 h of fasting and free access to water. The rabbits in both groups were physically examined for a number of traits on day 14. At 1, 7, and 14 days, blood samples were taken to check several biochemical indicators. These included the complete blood count, liver function, kidney function, and lipid profile. On day 14, similar to the control group, all rabbits in the therapy group had their vital organs removed for pathophysiological study ,22.The feed content ratio significantly impacts DF loading into the developed polymeric network. Drug loading (%) is associated with the swelling behavior of the polymeric network. Higher Na-Alg, CP-934P, and MAA contents resulted in higher loading capacities of the polymeric networks. DF loading (%) increased from 57% to 77% in formulations (SCH-1 to SCH-3) with variable Na-Alg contents, which was attributed to the low cross-linking tendencies inherent in the Na-Alg structure, which causes high swelling capacities and hence more significant drug loading. Also, at higher MAA and Na-Alg contents, the resulting viscosity and density can retain a higher drug load in the hydrogel . An incrSwelling studies evaluated the pH-responsive behavior of the developed hydrogel. Swelling studies revealed that constituents and feed ratios influence the hydrogels’ swelling behavior. All 12 formulations (SCH-1 to SCH-12) were analyzed for their pH-dependent behavior by placing them in buffers of 1.2 and 7.4 pH. At pH 1.2, all formulations exhibited minor swelling, whereas, at pH 7.4, significant and variable swelling (%) was demonstrated by all formulations.− groups promote electrostatic repulsion, due to which a reduction of the H-bond linkages within the chains occurs, resulting in the expansion of the polymeric chains and an influx of swelling media, and hence enhanced equilibrium swelling resulted.After increasing the content of Carbopol-934 P in the hydrogel (SCH-1 to SCH-3), an enhancement of the swelling from 455.55% to 537.26% was noted at basic pH of 7.4. Improved swelling at a higher CP-934P content may be ascribed to the high degree of ionization of the carboxylic groups of the CP at basic pH. The ionized anionic –COO− anions generated by the ionization of monomer and polymer at basic pH (9). Na-Alg has a lowly cross-linked structure, under which it has more swelling abilities. Higher Na-Alg concentration in the formulation thus expand more as compared to the formulations having lower concentrations. Afshar et al. (2020) have prepared ann Na-Alg/PVA-based hydrogel containing drug-loaded chitosan nanoparticles. The authors evaluated the equilibrium swelling% of the hydrogel and the effect of feed contents on its swelling%. Furthermore, they concluded that Na-Alg’s weak cross-linking tendencies presented better swelling behavior [Increasing the Na-Alg and MAA feed ratios in the formulations (SCH-4 to SCH-6 and SCH-7 to SCH-9) resulted in a more significant swelling of the developed hydrogels at pH 7.4. Na-Alg has a low cross-linking capability, thus resulting in more substantial swelling. By increasing the Na-Alg and MAA contents, the equilibrium swelling was increased from 535.18% to 743.19% and 421.25% to 478.56% in (SCH-4 to SCH-6) and (SCH-7-SCH-9), respectively, due to repulsion between –COOHigher MBA content in the formulations (SCH-10 to SCH-12) results in high cross-linking density, so the swelling was decreased from 550.70% to 311.65%. This reduction in swelling was attributed to the formation of a dense network that resists the penetration of swelling media, resulting in poor swelling. Similar findings were reported by Thakur and Arotiba (2018) in a study conducted on Na-Alg-co-AA hydrogels. They identified that the influence of MBA on the swelling ability follows a power law, i.e., Swelling capacity and MBA (cross-linker) content are inversely proportional . The resThe impact of ratios of various feed contents on the gel fraction is shown in −1. The ester group likely contributed to the C=O stretching seen at 1707.00 cm−1, whereas the ether group’s C–O–C stretching is observed at 1244.09 cm−1. At 798.53 and 619 cm−1, the C-Cl stretch reveals the existence of the halogen group [FTIR spectra of DF, Na-Alg, Carbopol-934P, MBA, and Na-Alg/CP co-poly (methacrylate) hydrogel were recorded and are shown in en group .−1 and 3078.39 cm−1 due to stretching movements of –OH and –CH2 groups, respectively. The peaks at 1523.16 cm−1 and 1705.07 cm−1 are related to the asymmetric and symmetric vibrations of the carboxylic groups. The peaks corresponding to the asymmetric stretching of the C–O–C groups appeared at 1101 cm−1, indicating glycosidic linkages of polysaccharides. A broader peak corresponding to the C–H deformation of mannuronic acid residue appeared at 827.46 cm−1 [In the IR spectrum of Na-Alg B, two ba.46 cm−1 .−1, 3493.09 cm−1, and 3342.60 cm−1 corresponding to –OH stretching movements. The peak that appeared at 2875.86 cm−1 was assigned to the stretching vibration of R–CH2 [−1 indicated a C=O stretching vibration, while a peak at 1246 cm−1 suggested a bending vibration of the –OH groups.The FTIR spectrum of Carbopol 934P C showed −1 and 3608.81 cm−1. A new peak appeared at 2586.54 cm−1 and was assigned to the stretching methylene cross-linker groups (MBA). A shift of the peaks corresponding to the C=O stretching occurred at 1737.86 cm−1. Our findings were similar to a study by Sarfraz et al. (2020) that evaluated and confirmed grafting in Carbopol-g-MAA-based nanogels [The FTIR spectrum of the developed Na-Alg/CP-co-poly(methacrylate) hydrogel D indicatnanogels .The DSC and TGA thermograms of pure DF, Na-Alg, CP-934P, and Na-Alg/CP-co-poly (methacrylate) hydrogels are shown in Around 245°C to 250 °C, DF decomposes, as seen in the significant weight loss (%) on the TGA thermogram A. A lossThe TGA thermography revealed a three-stage weight reduction for CP. The initial CP weight reduction of 8% might have been due to its losing moisture in the first step. During the second phase of weight loss, between 213 and 263 degrees Celsius, 15% of the weight was lost due to decarboxylation and the formation of unsaturated structures within polymer chains. After the third step, the CP began to depolymerize and break down, as shown by a 22.55 percent weight loss from 363 to 463 degrees Celsius C, until The TGA thermogram of the finished hydrogel revealed that the weight was lost in two distinct stages, at 220 degrees Celsius (12.61%) and 368 degrees Celsius (31.49%). At 500 degrees Celsius, the breakdown process began, leaving behind 38.78% of its original weight as residue D. This mThe TGA and DSC analysis results suggested that the developed hydrogel networks have improved thermal stability compared to their individual constituents.θ = 13.5°, 17.5°, 19.6°, 23.7°, and 27.45°, indicating its crystalline nature. The Na-Alginate diffractogram suggested the crystallinity of Na-Alginate as exhibited by intense peaks at 7.2°, 19.08°, 28.1°, 29.08°, 31.68° and 34.04°, whereas CP 934P showed much broader and more diffused peaks of low intensity, which indicate its amorphous character. The loaded hydrogel (formulation SCHL-9) showed broader peaks of low intensity, suggesting the successful incorporation of DF into the polymeric network, as shown in X-ray diffraction analysis was performed to evaluate the diffraction pattern and the effect of cross-linking on the crystallinity of DF. The pure DF diffractogram showed its characteristic peaks at 2Energy dispersive X-ray spectroscopy (EDX) was carried out to evaluate the elemental composition and the weight% of each detected element in the developed unloaded Na-Alg/CP-co-poly(methacrylate) hydrogel and the DF-loaded Na-Alg/CP-co-poly(methacrylate) hydrogel.The EDX emission spectrum of the unloaded hydrogel formulation SCH-9 indicated the presence of C, O, and Na as prominent peaks. In contrast, the loaded hydrogel spectrum revealed the presence of another Cl peak accompanied by a reduction of the O and Na peaks’ intensity. The Cl peak into the developed hydrogel suggested the uptake of the DF. According to the quantitative elemental analysis, diloxanide furoate was found to have 68.20% carbon, 20.12% oxygen, and 11.67% chlorine. According to A surface electron microscopy examination was used to examine the morphology of the hydrogels made from Na-Alg/CP-co-poly(methacrylate) (SCH-9). At 2500×, 5000×, and 10,000× magnifications, the surface morphology of the formed polymeric network was quite rough, as illustrated in The in-vitro drug release testing was done on all 12 formulations at pH 1.2 and 7.4. There was a significant discrepancy between the medicine release at acidic pH of 1.2 and basic pH of 7.4. Minimal DF release (less than 10%) has been seen at pH 1.2 across all formulations (SCH-1 to SCH-12). Due to the acidic pH, the carboxylic groups remained unionized. Consequently, releasing or biological media had trouble penetrating the networks due to the lack of polymeric chain expansion, and the DF was poorly dissolved. In solution, carboxylic acid groups are ionized into carboxylate ions at a pH of 7.4. These ionic species stimulate repulsive forces between the polymeric chains, resulting in the formation of spaces. The integrated moiety dissolves as the dissolving medium goes inward. Since there is a concentration gradient, the dissolved DF travels away from the center of the growing hydrogel. The hydrogels’ release profiles were profoundly affected by the relative amounts of polymer, monomer, and cross-linker used in their production. At pH 7.4, DF release was reduced when the concentration of CP-934 P in formulations was increased (SCH-1 to SCH-3). Hydrogels with a tight structure, such as those produced by a high concentration of carbopol, may be responsible for the reduced drug release. Limited drug release occurs because solvent penetration into such a dense network increases viscosity and shrinks micropores. Bera et al. (2015) developed Carbopol-based microbeads with Mucoadhesive potentials to facilitate the delivery of the anti-diabetic medication Glipizide. The same research group also found that CP promotes adhesiveness and inhibits medication release .The Na-Alg and MAA levels in formulations (SCH-3 to SCH-6 and SCH-7 to SCH-9) enhanced the diloxanide furoate release from 77.0 to 91.77 percent and from 77.08 to 84.77 percent, respectively .The increased swelling at higher Na-Alg and MAA concentrations is partly due to the massive number of pendant –COOH groups in the polymeric chains of these substances. –COOH group protonation and deprotonation significantly affect the release profile. The lack of repulsion between the pendent groups at acidic pH reduces the pore size, limiting drug release. This is because the protonation of COOH groups at an alkaline pH forms –COO– ions, which cause a significant release. Large pores formed due to electrostatic repulsion between the particles allow rapid medication release . The carThe DF release rate decreased from 81.63 to 67.85% when the MBA concentration in the synthesized formulations increased from SCH-10 to SCH-12. The enhanced cross-linking of the polymeric components with the increase in MBA concentration may account for this diminution. These results are consistent with the published literature ,44.2.” As can be shown in 2” than the first-order and Higuchi models for all formulations (SCH-1 to SCH-12).Mathematical models were applied to investigate and explain the in-vitro release kinetics of DF from the loaded hydrogel in phosphate buffer using the first order, zero order Korsemeyer Pappas, and Higuchi model. The mathematical model that best matches the release data was determined by calculating the coefficient of determination, or “RThis suggests that that the drug release from the gel network that occurs is controlled via pore diffusion, regardless of the drug concentration at the application site. The Korsmeyer Pappas model was applied to evaluate the drug release mechanism from the loaded hydrogels by using the release exponent “n”. The formulations SCH-3 to SCH-6 showed a value of n greater than 0.45 but less than 0.89, suggesting an anomalous (non-Fickian) diffusion of drug release. In contrast, all other formulations  represented super case II transport associated with values greater than 0.89. The case-II anomalous diffusion process is linked with controlled swelling.The assessment of the mucoadhesive force was done by increasing the weights on the right pan of the beam balance until the detachment of the two vial surfaces occurred.2) for all the formulations. The hydrogen bridging between the –COOH groups and the sialic acid residues of mucous contributes to the adherence of the developed hydrogel to the mucous membrane. Formulations developed with higher contents of MAA and MBA showed a reduction in the mucoadhesion force, attributed to bigger cross-linking, making fewer functional groups available for interaction with the mucin. The findings align with the authors’ results, revealing a direct relationship between the amounts of Na-Alg and the mucoadhesive strength of the developed microparticles [In the formulations SCH-1 to SCH-3 and SCH-4 to SCH-6, when increasing the concentration of CP-934P and Na-Alg, respectively, a rise in bioadhesion with the intestinal mucosa of rabbits was recorded. This may be because the prolonged contact of the hydrogel network in an aqueous medium resulted in the uncoiling of polymeric chains with subsequent penetration of these chains into the mucous. Several theories are quoted in the literature to explain the interaction of adhesive products with mucin, i.e., the electrostatic adsorption, wetting, diffusion, and fracture theories. The rinsing approach is adapted to determine mucoadhesive force . A highearticles .Incorporating a CP-934P, a synthetic polymer, into a hydrogel network renders high mechanical strength to the hydrogel due to its bioadhesive character. This inclusion also improves the toughness of the developed network, which can withstand the contractile forces of GIT .To determine the safety of the generated polymeric network, acute oral toxicity tests were performed. The rabbits were split into Group I (control) and Group II (treated group). Na-Alg/CP-co-poly(methacrylate) hydrogel-grounded particles with water assistance were delivered to the treatment group, whereas the control group received no treatment.We observed the animals for a total of 14 days to see whether the treatment altered their feeding patterns, behavior, or overall health in any way. There was a lot of drooling, crying and itchy skin, and a stuffy nose. Redness, inflammation, and tears were not seen in the treated animals. There were no unexplained deaths or changes in the treated group’s water or food intake.On day 14 of therapy, blood was collected from both groups for hematological and biochemical examination to determine whether or not the treatment impacted organ function. There was no discernible difference in the biochemical data between the treatment and control groups. On the 14th day after dosing, the animals from both groups were sacrificed, and their vital organs were removed to investigate any histo-pathological signs related to the treatment. The organs, such as the brain, liver, spleen, kidney, lungs, heart, and intestine, were preserved in formalin 10% solution and then examined microscopically for histopathology after being stained with H and E dyes. The gross morphology of the vital organs of the control and treated group is shown in The brain showed the presence of excessive glial cells with minimal shrinkage of the neurons. Overall, no signs of damage were seen in the axonal neurons. The heart tissue histology suggested no localized hypoxia, damage, or cardiomegaly. Only a few cells showed signs of morphological artifacts in some areas. The lung tissues in the microscope showed signs of hemorrhage visualized in the spleen tissue with normal alveolar wall thickness. There was no sign of alveolar edema. The kidney tissue appeared normal with a normal glomerulus and bowman capsule. The shape and appearance of the spleen were fine on the whole, with healthy-looking red and white pulp zones. Splenomegaly was not seen. Slight hepatocyte ballooning, indicative of liver fibrosis, is seen in Hydrogels based on Na-Alg/CP-934P-co-poly(MAA) were developed using a free radical polymerization approach, revealing themselves as a smart and environmentally sensitive network enabling pH-driven regulated distribution of DF to the colon. Adding Na-Alg and Carbopol-934P increased the material’s biodegradability, Mucoadhesion, and mechanical strength. The manufactured network’s increased toughness makes it an excellent choice for withstanding the contractile forces of GIT.


This research explored the clinical application of grade ≥ 3 infection predictive models for the newly diagnosed multiple myeloma (NDMM) population.It evaluated 306 patients with NDMM based on three different predictive models. The relationship between the grade ≥ 3 infection rates in NDMM and the scores was analyzed retrospectively. The cumulative incidence of early grade ≥ 3 infection was estimated using the Kaplan–Meier method and log-rank test to assess the statistical significance of the difference. To compare the predictive performance in the prediction of infection, the Receiver Operating Characteristic Curve (ROC) curve was used to show the area under the curve (AUC), and DeLong’s test was used to analyze the difference in AUC.p < 0.001; low-risk vs. moderate-risk vs. high-risk: 35.93% vs. 41.28% vs. 60.00%, p= 0.045; low-risk vs. moderate-risk vs. high-risk: 20.00% vs. 43.75% vs. 52.04%, p < 0.001). Statistical differences existed in the probability of early grade ≥ 3 infection among the different groups by the FIRST and IRMM scores but no statistical differences in the GEM-PETHEMA score . The FIRST score showed good discrimination and simple calculation with highest AUC. Further subgroup analysis showed that the FIRST score could still apply for patients treated with bortezomib-based regimen and frail patients.The incidence of grade ≥ 3 infection within the first 4 months of NDMM was 40.20%. Concerning the FIRST score , GEM-PETHEMA score , and Infection Risk model of Multiple Myeloma (IRMM) score , the probability of early grade ≥ 3 infection in the different groups showed statistically significant differences  is a simple and robust infection stratification tool for patients with NDMM and could be used in routine clinical work. However, no significant decrease has occurred in early mortality. Infection is a common early complication of newly diagnosed multiple myeloma (NDMM) and a major cause of early death. One study showed that 45% of early deaths within 6 months were due to infection . Some stp < 0.001) in the first 4 months. In 2022, the GEM-PETHEMA score . The results indicated that the IRMM model could predict early grade ≥ 3 infection. However, it has not been validated in the real world by other centers’ data. Validating IRMM scores with data from other facilities would also be logical.IRMM is an infection prediction model established in China using real-world data . This moTherefore, the purpose of this study was to determine which of these three prediction models can clearly recognize grade ≥ 3 infection in NDMM patients in real-world clinical practice, enabling clinicians to determine appropriate infection prevention strategies.2.2.1.A total of 306 patients with NDMM in the Third Hospital of Shanxi Medical University, First Hospital of Shanxi Medical University, and Shanxi Provincial People’s Hospital from May 1, 2016, to April 30, 2022, were enrolled in this retrospective study. All patients were diagnosed with MM using the International Myeloma Working Group (IMWG) criteria . The exc2.2.The clinical manifestations of the patient, typical imaging findings of infection, or the isolation of a microbial agent from peripheral blood or secretions in patients who also had concurrent clinical symptoms all served as the criteria for infection used in this study . The sevPatients were identified as nonfrail or frail based on their age, Charlson Comorbidity Index (CCI), and ECOG performance status : ≤ 75 ye2.3.Data were collected by studying medical records. The following factors were considered: age, gender, subtype, International Staging System (ISS) stage, Durie–Salmon (DS) stage, ECOG performance status, frailty assessment [the simplified frailty scale  was used2.4.The FIRST score, GEM-PETHEMA score, and IRMM score were employed to categorize enrollment populations. The values of the three predictive models and the grouping of risk levels are shown in 2.5.x ± s, and continuous data not conforming to a normal distribution expressed as medians (interquartile spacing). The continuous data were analyzed by analysis of variance, and the count data were analyzed by Chi-square test or Fisher exact probability method. The cumulative incidence of early grade ≥ 3 infection was estimated using the Kaplan–Meier method and log-rank test to assess the statistical significance of the difference. To compare the predictive performance in the prediction of infection, the ROC curve was used to show the area under the curve (AUC), and DeLong’s test was used to analyze the difference in AUC. Statistical significance was defined as a two-sided p-value of < 0.05.The data were analyzed using SPSS 26.0 statistical software, with count data expressed as frequencies and percentages (%), continuous data conforming to a normal distribution expressed as 3.3.1.A total of 306 patients with a median age of 64 years (IQR 57–71 years) were enrolled in the trial, comprising 172 men and 134 women. They included 144 IgG cases (47.06%), 70 IgA cases (22.88%), 65 light-chain cases (21.24%), 20 IgD cases (6.54%), 5 biclonal cases (1.63%), 1 IgM case (0.33%), and 1 oligosecretory case (0.33%). There were 12 cases (3.92%) of DS stage I, 29 cases (9.48%) of stage II, and 265 cases (86.60%) of stage III. There were 29 (9.48%) cases of ISS stage I, 113 (36.93%) cases of stage II, and 164 (53.59%) cases of stage III; 191 (62.42%) patients were in the frail group, and 115 (37.58%) were in the nonfrail group; 269 (87.91%) patients were treated with the bortezomib-based regimen, and 37 (12.09%) were treated with the non-bortezomib-based regimen. Patients receiving bortezomib-based therapy were routinely given drugs such as acyclovir or valacyclovir to prevent viral infections, but all patients were not used preventive antibacterial or fungal drugs. 3.2.Of the 306 patients with NDMM, 202 (66.01%) experienced 294 early infections (within 4 months). Of them, 75 patients had two or more infection events, and 151 (51.36%) infections took place in the first month . CTCAE gEscherichia coli was the most common Gram-negative organism, Enterococcus faecalis was the most common Gram-positive organism, and Candida albicans was the most common fungus. Classification and constituent ratios (%) of pathogens in patients with NDMM are shown in The most common sites of early grade ≥ 3 infections were the lower respiratory tract , upper respiratory tract , and gastrointestinal tract . There were 59 bacterial infections, 20 viral infections, and 9 fungal infections . 3.3.p < 0.001), and the probability of early grade ≥ 3 infection was significantly higher in the high-risk group than in the low-risk group  and a high-risk group . The difference in grade ≥ 3 infection between the two groups was statistically significant . The ORs (95% CI) for the high-risk/low-risk group, moderate-risk/low-risk group, and high-risk + moderate-risk/low-risk group were 2.675 (1.207–5.929), 1.254 (0.764–2.058), and 1.478 , a moderate-risk group , and a high-risk group . The probability of early grade ≥ 3 infection in different stratifications showed statistically significant differences . The ORs (95% CI) for the high-risk/low-risk group, moderate-risk/low-risk group, and high-risk + moderate-risk/low-risk group were 4.340 (2.207–8.534), 3.111 (1.625–5.957), 3.597 , a moderate-risk group , and a high-risk group . The probability of early grade ≥ 3 infection in different stratifications showed a statistically significant difference . The high-risk and moderate-risk groups were statistically different than the low-risk group . However, the difference between the high-and moderate-risk groups was not significant . The OR (95% CI) for the high-risk group/low-risk group was 2.901  were treated with the bortezomib-based regimen and 37 (12.09%) with the non-bortezomib-based regimen. Based on the FIRST score, patients treated with the bortezomib-based regimen (269 cases) were divided into a low-risk group  and a high-risk group . The high-risk group had a significantly higher probability of early grade ≥ 3 infection than the low-risk group . The OR (95% CI) for the high-risk group/low-risk group was 2.728  patients were in the frail group, and 115 (37.58%) were in the nonfrail group. According to the FIRST score, frail patients were divided into a low-risk group  and a high-risk group . The probability of early grade ≥ 3 infection was significantly higher in the high-risk group than in the low-risk group  in a real-world population of patients with NDMM (mostly frail). According to our studies, the FIRST score and IRMM score could accurately predict grade ≥ 3 infection. However, the FIRST score, which comprises ECOG, β2-microglobulin, hemoglobin, and lactate dehydrogenase, is a simple and valuable infection classificatory system for patients with NDMM that could be employed in regular clinical use. The FIRST score is also suitable for frail patients and those taking bortezomib-based therapy.Our results show that 66.01% (202/306) of NDMM patients reported infections within the first 4 months from the initiation of treatment, and 40.20% (123/306) ≥ 3 TE infections. The total number of any grade infections in this study was higher than the results of other series . The GEM-PETHEMA score performed poorly in infection prediction and group discrimination. Based on real-world data, statistical differences existed in the incidence of infection between the high-risk group and the low-risk group  by GEM-PETHEMA score, but with no statistical difference between the moderate-risk group and the low-risk group  or between the high-risk group and the moderate-risk group . Furthermore, the cumulative incidence of infection did not differ statistically between the three GEM-PETHEMA score groups (p = 0.090). This indicates that the GEM-PETHEMA score does not appear to have effective discrimination for grade ≥ 3 infection in the real world. When the GEM-PETHEMA score research group tested the FIRST score using their data, they found no significant association with a higher risk of early severe infection between the high-risk and low-risk groups  and between the moderate-risk and low-risk groups (p < 0.001). However, the differentiation between the high-risk and moderate-risk groups was unclear (p = 0.437).Besides the uniform clinical trials, a robust risk stratification system should work effectively in a wide range of clinical circumstances. This study demonstrates that the FIRST score can predict grade ≥ 3 early infection in the real world and shows reasonable distinction. The infection rate of the high-risk group was significantly higher than that of the low-risk group when divided by FIRST score , as well as the sensitivity and specificity of the three models (p > 0.05). However, the FIRST score model was only slightly better (AUC: 0.650 vs. 0.619 vs. 0.630). In addition, the cumulative infection rate by the FIRST and IRMM scores in the first 4 months was significantly statistically different, whereas the GEM-PETHEMA score showed no significant difference. Additionally, considering the prediction indicators and calculation process, the calculation of the IRMM score is relatively complex, whereas the calculation of FIRST and GEM-PETHEMA scores is relatively simple. Combining validated results and computational processes, we believe that the FIRST score will be more beneficial in clinical practice.This study attempted to further analyze these three models as to which may have a better ability to distinguish grade ≥ 3 infection in NDMM patients using ROC curves and the Youden index. However, from the validation results, the three prediction models were not much different. The probability of grade ≥ 3 infection in different infection groups was compared (However, notably, induction regimens for developing the FIRST score did include non-proteasome-based regimens. In the real world, especially in China, most patients are treated with bortezomib-based regimens . Our resMonitoring high-risk patients and taking active relevant preventive measures may be significant. The indications for antimicrobial prophylaxis are controversial. A randomized phase III URCC/ECOG study showed that the use of prophylactic antibiotics during the first 2 months of treatment did not reduce the incidence of severe infection . In contOur study has several limitations. First, despite multicenter data collection, the sample size was relatively small. The nature of retrospective design narrows the conclusion, so biases were unpreventable. Second, the regimen containing daratumumab has been recommended for the first-line treatment of NDMM . HoweverIn conclusion, our study confirms that the FIRST score is a simple and robust infection stratification tool for patients with NDMM, including frail patients and those being treated with bortezomib-based therapy. It can discriminate patients with grade ≥ 3 infection, help to guide infection prevention, and improve outcomes of patients with NDMM.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Shanxi Bethune hospital ethics committee. Written informed consent for participation was waived for this study.WT and JW designed and supervised the work, interpreted the results, and approved the final version. XL, WL, LZ, LY, QY, JZ, SH, and XC collected clinical data. XL and XC analyzed data. XL, WL, WT, and JW wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.